Development of inhibitory immune responses to vector and transgene products continues to be a major impediment to long-term correction of genetic disorders. To circumvent this problem, we propose to deliver and express genes early in the maturation of the immune system, with the goal of achieving tolerance to therapeutic gene products and gene delivery vectors. This would also enable pre-emptive treatment of genetic Disorders with perinatal morbidity and mortality. In this context, one of the important differences in ontogeny between mouse and human immune systems is that T cell maturation primarily occurs early during the postnatal period in the mouse, but during mid-gestation in human development. Thus, the'immunological maturity of postnatal mice within the first 3 weeks of life represents a more relevant preclinical model to test strategies for perinatal gene delivery and induction of immunotolerance in humans. Therefore, the overall goal of this revised proposal is to develop strategies for induction of immunotolerance to AAV vector- mediated delivery and expression of transgenes in neonatal mice. Throughout, we will (1) compare bio- distribution, level, and duration of gene expression after primary neonatal delivery and secondary adult re- challenge with highly efficient AAV 1 and 8 serotype vectors, (2) monitor immune responses to the vectors and an intracellular reporter, luciferase, or a secreted therapeutic protein, Factor IX, and (3) define the critical period in the first 3 weeks of life when induction of tolerance is feasible. We will also examine whether differences in AAV 1 vs. 8 tissue tropism affect immune response, and whether the route of administration (IV or IP delivery) affects immune responses to human Factor IX in normal and hemophilic animals. Finally, through adoptive transfer and immunodepletion experiments, we will identify specific immune mechanisms that may influence the persistence or abrogation of gene expression following neonatal gene delivery and adult re-challenge with AAV-murine F.IX vectors in hemophilic animals. RELEVANCE. These studies will define a period in immune ontogeny when viral gene delivery vectors can be repeatedly injected and expressed without eliciting inhibitory immune responses that limit therapeutic gene expression. Furthermore, for many genetic disorders, intervention in the perinatal period to correct the disorder would limit or abrogate the development of irrevocable, often lethal consequences of the disease.